Nitrate, the most abundant combined, dissolved form of inorganic nitrogen in global oceans, is a common source of nitrogen (N) for phytoplankton including cyanobacteria. Using a nested polymerase chain reaction (PCR) method, the diversity of the cyanobacterial nitrate reductase gene, narB, was examined in plankton samples from a variety of marine habitats. A total of 480 narB gene fragment sequences were obtained from a coastal coral reef (Heron Island, Australia), open-ocean tropical and subtropical oceanic waters (Atlantic and Pacific Oceans) and a temperate N. Pacific Ocean site (34 degrees N, 129 degrees W). Phylogenetic analyses distinguished eight picocyanobacterial narB clades comprised of DNA sequences derived from the nutrient-replete coastal, nutrient-deplete pelagic and tidally influenced coral reef habitats. The phylogeny of recovered narB gene sequences was consistent with 16S rRNA and ITS sequence phylogenies, suggesting minimal horizontal gene transfer of the narB gene. Depending on sampled habitat, environmental narB sequence types segregated into three divisions: non-picocyanobacterial, coastal picocyanobacterial and open-ocean picocyanobacterial sequences. Using a reverse transcription PCR method, narB mRNA sequences were amplified from Heron Island samples, indicating that narB expression can be detected in environmental samples.